JIA-2018-09

2033 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 the 5´-terminus of ABD2 at the Knp I site. The microtubules marker protein (mCherry-MAP65-1) and ER marker protein (mCherry-HDEL) were kindly provided by Prof. Tao Xiaorong from Nanjing Agricultural University, China. Seeds of N . benthamiana were germinated for 1 week at 22°C in a constant temperature incubator, and 2 weeks later the seedlings were transplanted and grown in a greenhouse under a 12 h light and 12 h dark cycle at 22°C with routine watering and fertilization. After 3–5 weeks when N . benthamiana reached the ten leaf stage, the upper emerged leaves were used for Agrobacterium infiltration. 2.2. Transient infiltration and DAPI staining Agrobacterium transformed with SVBV ORFs or P6 mutants was diluted to OD=1.0 with MMA buffer (10 mmoL L –1 MgCl 2 , 10 mmoL L –1 MES (pH 5.6) and 100 μmoL L –1 acetosyringone). After 2–3 h, N . benthamiana leaves were agroinfiltrated with Agrobacterium strains harboring the SVBV derivatives with ORFs or P6 mutants, respectively. For co-infiltrations with P1 and labeled P6 or P1 and the P6 mutant combinations, Agrobacterium strains were mixed at ratios of 3:5 respectively. For visualization of nuclei, lower epidermal N . benthamiana leaf strips were excised on a microscopic slide, and then the unfold epidermis was soaked in 1 μg mL –1 DAPI for 15 min with gently shaking. The stained epidermis was washed with water six times and visualized by confocal microscopy. 2.3. Confocal microscopy Agroinfiltrated N . benthamiana leaf strips were mounted in water under a coverslip, and images were acquired on an OLYMPUS FluoView™ FV1000 Confocal Imaging System (Hamburg, Germany). RFP was excited at 525 nm and images were captured at 598 nm. GFP was excited at 488 nm and captured at 522 nm. For double labeling experiments, the green and red channels were imaged separately and then superimposed. For time-lapse microscopy, images were obtained every 6 or 10 s from a single optical plane. Images were processed using Adobe Photoshop Software. 2.4. RNA and protein extraction Leaves co-infiltrated with P6 or P6 mutants and GFP were harvested at 3 days post infiltration (dpi). Total RNA was extracted from leaf tissue using Easy Pure Plant RNA Kit (Tiangen, China). Agarose gel electrophoresis was used to assess the RNA quality, and samples with minimal RNA degradation were used for Northern blot analysis. Proteins were extracted from leaf samples with 3 mL PBS extraction buffer (137 mmoL L –1 NaCl, 2.7 mmoL L –1 KCl, 10 mmoL L –1 Na 2 HPO 4 , 2 mmoL L –1 KH 2 PO 4 ) per gram fresh leaf material. Insoluble materials were separated by centrifugation (10 min, 12 000×g, 4°C) and the lysate was collected for Western blot analysis. 2.5. Northern blot analysis Equal amounts of RNA (5 μg) mixed with an equal-volume of 2× loading buffer were loaded on gel lanes and separated in a 1.0% agarose gel. The RNA was transferred to a nylon membrane (Amersham, Little Chalfont, UK) in 20× SSC. GFP RNA probes were amplified by PCR with specific primers and then labeled with DIG (Labeling and Detection Starter Kit II, Roche, Switzerland). For labeling reactions, 1 μg of purified GFP DNA was mixed with 4 μL DIG-HIGH prime in a 20-μL reaction system at 37°C for 20 h. Prehybridization, hybridization, washing and detection of hybridizing bands were carried out according to the manufacturer’s protocol of Labeling and Detection Starter Kit II (Roche, Switzerland). 2.6. Western blot analysis For GFP detection, 30 μL lysate was loaded into SDS-PAGE to separate total proteins and the proteins were transferred to a Pure Nitrocellulose Blotting Membrane (PALL) with Trans-Blot SD Semi-Dry Transfer Cell 170-3940 (Bio-Rad, USA) at 15 V for 15 min. GFP antibody of Boster Biological Technology was used at 1:5000 dilution. The membranes were stained with BCIP/NBT. 3. Results 3.1. The SVBV P6-GFP fusion protein forms IBs IBs have been found in many viruses-infected cells and their components provide important clues about replication, movement and host defense (Novoa et al . 2005; Wileman 2006; Netherton et al . 2007; Moshe and Gorovits 2012). The P6 protein and icosahedral virus particles are the predominant IBs constituents in CaMV-infected cells (Baughman et al . 1988). To determine which SVBV-encoded proteins form IBs, we fused GFP to the carboxy-terminus (C-terminus) of each SVBV ORF, and the corresponding constructs were transformed into Agrobacterium tumefaciens and agroinfiltrated into N . benthamiana leaves. Subsequently, the agroinfiltrated areas were examined by laser scanning confocal microscope at 3 dpi. The SVBV-GFP proteins varied in their cytoplasmic and nuclear distribution. P1-GFP formed foci that were distributed along the cell boundary, which is similar to the